ABSTRACT Diabetes mellitus (DM) is a widespread metabolic disorder of glucose homeostasis characterized by an absolute or relative deficiency in insulin production. Endoplasmic reticulum (ER) dysfunction, particularly unregulated ER stress, is a key mediator of ?-cell pathology that causes a decline in both ?-cell mass and ?- cell function. Accordingly, genetic defects in the molecular machinery responsible for maintaining ER homeostasis result in monogenic forms of diabetes, such as Wolfram syndrome. This autosomal recessive, progressive neurodegenerative disorder stems from mutation of the WFS1 locus, which manifests clinically first as juvenile-onset diabetes, secondary to ER stress-mediated ?-cell dysfunction. Our preliminary data indicate that ?-cells depleted of WFS1 exhibit impaired insulin secretion and reduced insulin content. Further, our data demonstrate that knockdown of WFS1 is associated with decreased ER Ca2+, increased cytosolic Ca2+ and increased ?-cell death. Most notably, our pilot studies indicate that increasing WFS1 expression in vitro increases insulin production, thereby suggesting a protective role for WFS1 in ?-cells against metabolic challenges such as high glucose. This proposal therefore seeks to functionally characterize the role of WFS1 in pancreatic ?-cell viability and ?-cell function by evaluating the hypothesis that WFS1 regulates insulin production and secretion through the regulation of key ER homeostasis molecules and ER Ca2+ transporters. To address this hypothesis, this proposal seeks to determine the role of WFS1 in ?-cells under diet-induced metabolic stress through the study of conditional ?-cell-specific WFS1 overexpression mice on a high fat diet (Aim 1). The mechanism(s) of WFS1-mediated insulin regulation will be investigated in vitro. This proposal also seeks to determine the mechanism(s) of WFS1-mediated Ca2+ regulation in ?-cells by evaluating the stability of major ER Ca2+ transporters in the context of WFS1 expression (Aim 2). Identification and functional characterization of WFS1 alleles that disrupt ER Ca2+ will be further investigated using high throughput functional assays of WFS1 mutation libraries. In this manner, this proposal aims to evaluate the therapeutic potential of WFS1 and WFS1-mediated insulin regulation pathways in the context of diet-induced diabetes, while also characterizing WFS1 mutations in the context of ER Ca2+ homeostasis. This research therefore holds the potential to capitalize on a prototype of ER stress-mediated disease to expand our understanding of the mechanisms by which ER dysfunction triggers ?-cell pathology in more common forms of diabetes, such as type 2 DM.